RNA-Seq: differential expression analysis of controls
Libraries required
Load salmon object
Filtering gene counts
Function
Counts from all data
Differeitial analysis using limma
SVA
se <- SummarizedExperiment(assays = counts.filt)
se@colData <- DataFrame(salmon@phenoData)
sv.pl <- svacor(SE = se, form = ~ 0 + Group)## Using variance-stabilizing transformation
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
## Number of significant surrogate variables is: 3
## Iteration (out of 5 ):1 2 3 4 5
limma fit
design <- model.matrix(~ 0 + SV1 + SV2 + SV3 + Group, data = sv.pl@colData)
colnames(design) <- gsub(pattern = "Group", replacement = "", x = colnames(design))
dds <- calcNormFactors(DGEList(counts = counts.filt))
v <- voom(dds, design = design)
contrast.matrix <- makeContrasts(PND15 - PND8, Adult - PND15, levels = design)
fit <- lmFit(v)
fit2 <- contrasts.fit(fit, contrast.matrix)
fit2 <- eBayes(fit2)DEA results
pnd8.pnd15 <- as.data.frame(topTable(fit2, coef = 1, number = Inf))
pnd8.pnd15 <- data.frame(Genes = rownames(pnd8.pnd15), pnd8.pnd15, stringsAsFactors = F)
pnd15.adult <- as.data.frame(topTable(fit2, coef = 2, number = Inf))
pnd15.adult <- data.frame(Genes = rownames(pnd15.adult), pnd15.adult, stringsAsFactors = F)vst Counts data and pData
dge <- DGEList(counts = counts.filt, group = salmon@phenoData$Group)
dge <- calcNormFactors(object = dge, method = "TMM")
cpm <- cpm(dge, log = T)
cpm <- removeBatchEffect(x = cpm, covariates = design[, 1:3], design = design[, 4:6])
data <- list(cpm = cpm, vstSV = sv.pl@assays[[2]], voomE = v$E, pData = salmon@phenoData)
save(data,
file = "./output/data_pData.RData", compress = T,
compression_level = 3
)Results
dea.list <- list(
`PND8 vs PND15` = as.DEA(pnd8.pnd15),
`PND15 vs Adult` = as.DEA(pnd15.adult)
)
normCounts <- v$E
voomEList <- v
dea.limma <- list(
`PND8 vs PND15` = pnd8.pnd15,
`PND15 vs Adult` = pnd15.adult
)Save RData files
save(dea.list,
file = "./output/dea_SC_Controls.DEA.RData", compress = T,
compression_level = 3
)
save(normCounts,
file = "./output/normCounts_voom_SC_Controls.RData", compress = T,
compression_level = 3
)
save(voomEList,
file = "./output/voom_EList_SC_Controls.RData", compress = T,
compression_level = 3
)
save(dea.limma,
file = "./output/limma_SC_Controls.RData", compress = T,
compression_level = 3
)PCA plots
Raw counts filtered
SVA (VST)
SessionInfo
## ─ Session info ──────────────────────────────────────────────────────────
## setting value
## version R version 3.6.1 (2019-07-05)
## os Ubuntu 16.04.6 LTS
## system x86_64, linux-gnu
## ui X11
## language (EN)
## collate en_US.UTF-8
## ctype en_US.UTF-8
## tz Europe/Zurich
## date 2019-09-02
##
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